Juvenile forms of severe and recurrent rhabdomyolysis are inherited disorders characterized by the presence of myoglobinuria, serum creatinine kinase levels>10,000 IU/L and acute kidney injury [1, 2]. The precise incidence is still debated since this clinical entity is often unknown to physicians, but it is thought to represent 10-15% of the whole rhabdomyolysis in childhood. Its prognosis is poor notably because both the severity and the occurrence of further exacerbations are unpredictable [3] and the treatments remain symptomatic. Lipin-1 disease is one cause of recurrent and severe rhabdomyolysis occurring in infancy (Zeharia et al., 2008; Michot et al., 2010; Michot et al., 2012). Half of the patients die during rhabdomyolysis bouts (Bergounioux et al., 2012).
Accordingly, there is a need to develop new drugs for treating rhabdomyolysis. In this way, it has been suggested that characterization of new compounds for treatment of rhabdomyolysis may be highly desirable.
Therefore there is a crucial need to identify the pathogenic mechanisms involved in rhabdomyolysis flares, although some of them have been already characterized. Indeed, the clinical spectrum of severe and recurrent juvenile rhabdomyolysis appears quite heterogeneous regarding the nature of the different diseases. However they share a common mechanism responsible for myoglobinuria: these defects lead to an impairment in adenosine triphosphate metabolism and to a deregulation of Na+/K+ or Na+/Ca2+ ion channels and finally to a rapid increase of cytoplasmic ionized calcium concentration that leads to the destruction of the muscle cell [1, 4, 5].
Inherited rhabdomyolyses are precipitated and worsened by febrile illness or exercise, two conditions well known to be associated with high circulating levels of pro-inflammatory mediators such as cytokines and chemokines [6-8] and high temperature. Also, the inventors recently showed that rhabdomyolysis due to aldolase A deficiency involves an exacerbation of enzyme deficiency at high temperatures [9].
Potential links between metabolic diseases and chronic inflammation have already been reported in various contexts such as type 2 diabetes, obesity or atherosclerosis [6-8] leading to the proposition of immunometabolism as an emerging frontier [10]. In type 2 diabetes, various circulating inflammatory mediators have been detected such as Tumor Necrosis Factor (TNF), interleukins (IL) or adipokines. Attention has recently shifted to IL-1β which is now incriminated as one of the main pathogenic actor, by activating the c-Jun NH-(2)-terminal kinase (JNK) and triggering serine phosphorylation of insulin receptor substrate-1 (IRS1). IL-1β impairs the insulin-Phosphatidylinositol-3 kinase (PI3K)-Akt signaling pathway in insulin-sensitive tissues contributing to insulin resistance. Regarding inherited metabolic disorders, inflammation strongly contributes to the pathogenesis of Majeed syndrome [11, 12] or Mevalonate Kinase deficiency [13] that both belong to the clinical spectrum of auto-inflammatory disorders [14, 15]. Majeed syndrome is due to LPIN2 defect, a homologue of LPIN1, which controls triacylglycerol (TAG) synthesis, JNK/AP-1 (Jun N-terminal kinase and transcription factor AP-1) pathway activation and ultimately the up-regulation of pro-inflammatory genes under palmitic stimulation [14]. As direct evidence, biotherapies targeting the IL-1-related pathways have improved the outcomes of these two conditions. Of interest, some inherited metabolic disorders have reported a direct toxicity of inflammatory mediators in target organs, as this is the case for neurotoxicity of glutamate that, in type 1 glutaric aciduria, is potentiated by TNF-α [16-19].
The picture is less clear regarding skeletal muscle. Despite clear clinical evidence for its presence, no report has investigated the role of inflammation during the course of severe and recurrent juvenile rhabdomyolysis so that important questions remain to be addressed. The inventors and others reported lipin-1 (LPIN1) mutations as a cause of severe and recurrent rhabdomyolysis triggered by febrile illness or exercise [20-24]. Lipin-1 (phosphatidic acid phosphatase 1, PAP1, EC 3.1.3.4) dephosphorylates phosphatidic acid (PA) to DiAcylGlycerol (DAG), a common precursor for triglycerides (TAG) and phospholipid (PL) synthesis [25, 26]. It also has transcriptional co-regulator activity, which, through association with PPARα, PGC-1α, SREBP1 or NFATc4, regulates lipid metabolism and the mitochondrial respiratory chain [27-30]. Primary myoblasts from patients exhibit a dramatic decrease in LPIN1 expression and PAP1 activity, associated with a significant accumulation of lipid droplets (LD) [31]. This lipid myopathy has been explained in partly by an overexpression of ACACB, the gene encoding acetyl-CoA carboxylase beta, a key enzyme in the fatty acid synthesis/oxidation balance, that is associated with a decrease of Carnitine PalmitoylTransferase I (CPT1) activity in the presence of pro-inflammatory conditions (TNF-α+IL-1ß) [31]. Lpin1fld/fld mice also present similar muscle lipid accumulation, recently described as the consequence of a blockade in autophagic flux and accumulation of aberrant mitochondria [32].
The inventors demonstrated that the pathogenic mechanism of rhabdomyolysis in lipin-1-deficient patients combines the predisposing constitutive impairment of lipid metabolism and its exacerbation by pro-inflammatory cytokines; crucial questions on the existence of a deregulation in the inflammatory responses and their underlying mechanisms remained. Accordingly, lipins are expressed in human and other species' macrophages [14, 33-35], and lipin-1 has been suggested to be an anti-inflammatory enzyme by repressing Nuclear Factor of Activated T-Cells Cytoplasmic, Calcineurin-Dependent (NFATc)-4 [28] and Monocyte Chemoattractant Protein (MCP)-1 activities [36].
The innate immune system acts as the first line of defense against invading pathogens. Induction of antiviral innate immune responses depends on a family of innate immune receptors, the Toll-like receptors (TLR) which detect “danger signals”, activate signaling pathways and induce inflammatory responses [37, 38, 39]. TLRs are present either at the cell surface or in endosomal compartments, TLR9 being an example for the latter case [40]. Importantly, a new TLR transduction mechanism impinges directly on Ca2+ transfer [41], the final step responsive for rhabdomyolysis.
A membrane lipid network important for the regulation of TLR activation and signaling has been recently described {Koberlin, 2015 #2452} {Kano, 2011 #2143}. In the same way, the regulation of Ras-related in brain (Rab) proteins, that tightly control the innate immune system {Jahn, 1999 #2154}, is dependent of their immediate lipid microenvironment [43]. Rab proteins regulate the trafficking between the different subcellular membranous compartments of eukaryotic cells [42]. A Rab7 like protein called Rab7b, also localized on late endosomes and lysosomes, and mainly expressed in innate immune cells and muscle, has recently been involved in retrograde protein transport [43-45]. Rab7b downregulates TLR9 and TLR4 by regulating the transport from late endosomes to the trans golgi network {Yao, 2009 #2062; Wang, 2007 #2063; Ge, 2014 #2155; {Bucci, 2010 #2058; Klaver, 2015 #2372}. Recently an additional role for Rab7b has been described in skeletal muscle in the control of actin remodeling [46]. The phospholipid compositions of the different subcellular organelles differ from each other [47], notably one of the characteristic features of early endosomes is their high content of phosphatidylinositol 3-Phosphate (PI3P class III)[48, 49]. PI3P has received a lot of attention with respect to endocytosis and signal transduction via endosomes [49-53, 54{Gillooly, 2003 #2294, 55], notably in skeletal muscle [56]. Remarkably, Phosphatidic Acid Phosphatases (PAP) play a role in this lipid environment, as the knock-down of Pah1 activity, the single yeast homologue of the Lipin family, has been involved in the decrease of the PI-3-kinase Vps34p and of its product PI3P. This results in a reduction of Rab proteins including Rab Ypt7p, Vps34p and Vps39p, a subunit of the HOPS (homotypic fusion and vacuole protein sorting) tethering complex, which are required for SNARE activity and vacuole fusion [57]. Zhang et al demonstrated that lipin-1 PAP activity defect lead to autophagy blockade by decreasing the PKD-Vps34 cascade [32] in rhabdomyolysis statin-induced in LPIN1fld/fld mice.
The inventors investigated innate immunity in lipin-1 patients, and showed an excessive release of pro-inflammatory mediators, which was explained by a hyper-sensitivity of immune and non-immune cells to TLR-9 ligands, the latter phenomenon resulting from an alteration in the endosomal PI3P content and architecture, and a down-regulation of Rab7b responsive for a blockage of TLR9 retrograde transport. Hyperactivity of TLR9 leads to an increase of intra-cellular calcium, responsive for skeletal muscle lysis. This allowed the inventors to propose calcium flux investigation as an original method to study Lipin-1 disease and appropriate treatments, and to consider TLR9 antagonists or inhibitors and anti-calcic drugs to prevent further relapses in patients.
There is no previous disclosure in the art of the role of TLR9 in rhabdomyolysis, and the use of TLR9 antagonists and inhibitors of TLR9 expression in the treatment of rhabdomyolysis.